Enigma-Mdm2 interaction and uses thereof

ABSTRACT

The present invention relates to Enigma (PDLIM7)-Mdm2 interaction and use thereof. More particularly, it may induce an effective apoptosis of cancer cells by inhibition of an Enigma expression or an Enigma activity which induces Mdm2 destabilization and p53 activity; it may assess the prognosis of anti-cancer therapy by determining that Enigma, which is induced by SRF, is overexpressed in cancer tissues with Mdm2; it may screen anti-cancer activity substances by selecting a factor to inhibit specific binding between Enigma and Mdm2. Enigma-Mdm2 interaction and Enigma expression regulation may be utilized usefully for preventing cancers and developing therapeutic methods and anti-cancer agents.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 12/704,226,filed Feb. 11, 2010, now U.S. Pat. No. 8,088,750, issued Jan. 3, 2012,which claims the benefit of priority from Korean Patent Application No.10-2009-0023510, filed on Mar. 19, 2009, both of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to Enigma (PDLIM7)-Mdm2 interaction anduse thereof, more particularly, to a method of treating cancer usinginteraction between Enigma (PDLIM7) and Mdm2, and regulation of Enigmaexpression, and an anti-cancer agent.

2. Description of the Related Art

The tumor suppressor p53, which regulates the genes associated with cellgrowth arrest and apoptosis, has been known as an essential factorpreventing normal cells from becoming cancerous cells by removingabnormal cells (Michael and Oren, Cncer Biol., 13, 49-58, 2003). Thisfunction of p53 is very strictly regulated for the survival of normalcells, and Hdm2/Mdm2 (human/mouse double minute 2; hereinafter referredto as “Mdm2”) plays a main role in the regulation. The Mdm2, which is anE3 ubiquitin ligase that ubquiquinates p53 for degradation, regulatesprotein level of p53 in the cell (Haupt et al., Nature, 387, 296-299,1997; Honda et al., FEBS Lett., 420,25-27,1997). Furthermore, Mdm2 formsan autoregulatory loop where the Mdm2 expression is regulated by thetranscriptional control of p53 (Michael and Oren, Cancer Biol., 13,49-58, 2003). Also, the Mdm2 ubiquitinates Mdm2 itself for degradation(Fang et al., J. Biol. Chem., 275, 8945-8951, 2000; Honda and Yasuda,Oncogene, 19, 4173-4176, 2000).

The Mdm2 deactivates p53 or acts p53-independently as an oncogene and isactually overexpressed throughout various human cancer tissues (Onel, K& Cordon-Cardo, C. Mol Cancer Res 2, 1-8, 2004). Since a negativeregulation of the Mdm2 function in a cancer cell with a wild type p53may induce apoptosis of the cancer cell, Mdm2 has been a useful targetfor the development of anti-cancer agent (Onel, K & Cordon-Cardo, C. MolCancer Res 2, 1-8, 2004). However, the apoptosis resulting from p53stabilization through the regulation of the Mdm2 function may causeserious side effects in normal cells as well as in cancer cells, andtherefore the elucidation of a pathway in cancer cells is urgentlyneeded to regulate Mdm2 selectively.

PDLIM7 protein Enigma has PDZ and LIM domains (Bach, Mech.Dev. 91, 5-17,2000). The LIM domain of the Enigma consists of three zinc fingers andbinds with protein kinases or factors associated with insulin signaling,etc. (Kuroda et al., J. Biol. Chem., 271, 31029-31032,1996; Wu et al.,J. Biol. Chem., 271, 15934-15941,1994). Furthermore, Enigma binds withRet/ptc associated with thyroid cancer to activate it (Durick et al.,Mol. Cell. Biol., 18, 2298-2308, 1998). However, Enigma's function in acell has not been clearly understood.

The factors such as YY1, Gankyrin, Daxx, etc. are known to stabilizeMdm2 and therefore to weaken the activity of p53. The YY1 binds withboth Mdm2 and p53 to thereby promote the degradation of p53, andGankyrin enhances the enzyme activity of Mdm2 to thereby promote thedegradation of p53 (Sui et al., Cell, 117,859-872,2004; Higashitsuji etal., Cancer cell, 8,75-87, 2005). While the Daxx binds with Mdm2 tostabilize Mdm2 and inhibit p53, it does not affect theself-ubiquitination activity of Mdm2 (Tang et al., Nat. Cell. Biol.,8,855-862, 2006). Mdm2 expression increases as cells proliferate (Fenget al., J. Biol. Chem., 279, 35510-35517, 2004), and however, how thisoccurs is not clear.

Hence, as a result of investigation on Enigma functions, the presentinventors revealed a novel Mdm2-p53 regulatory mechanism which isclearly distinct from the typical regulatory factors for an Mdm2-p53pathway in that Enigma specifically inhibits the self-ubiquitination ofMdm2 by Mdm2-dependently binding to p53, and promotes the degradation ofp53 by enhancing Mdm2-dependent p53 ubiquitination. Additionally, firstrevealed was that the conditions of cellular proliferation, and theexistence of SRF-Enigma-Mdm2 pathway in human liver cancer and stomachcancer tissues may weaken p53. Also revealed was that the Enigmaoverexpression in a cell increases the cell survival ability and inducesits resistance to anti-cancer agents. Thus, the present invention comesto completion by revealing that p53 can be activated by inhibiting theoverexpression of Enigma in cancer cells, that anti-cancer agents can bescreened by selecting inhibiting factors of the Enigma-Mdm2 interaction,and that identifying the Enigma expression level in treatment withanti-cancer agents maybe used for the identification of the anti-cancereffect.

SUMMARY OF THE INVENTION

An object of the present invention is to elucidate relationships betweencellular function of Enigma and cancer initiation and progression, toprovide a method and formulation to induce apoptosis of cancer cells byinhibiting Enigma expression, and to provide a method of screeningcandidate anti-cancer substances, using the interaction between Enigmaand Mdm2 or the expression regulation of Mdm2 and p53 by Enigma.

In order to achieve the objects, the present invention provides ananti-cancer composition including Enigma expression or activityinhibitor as an effective ingredient.

The present invention also provides an anti-cancer adjuvant includingEnigma expression or activity inhibitor as an effective ingredient.

The present invention also provides a method of treating cancer,including administrating an inhibitor of Enigma expression or activityin a pharmaceutically effective amount to an individual suffering fromcancer.

The present invention also provides a method of preventing cancer,including administrating an inhibitor of Enigma expression or activityin a pharmaceutically effective amount to an individual.

The present invention also provides a method of decreasing Mdm2stability and increase p53 activity by inhibiting Enigma expression oractivity.

The present invention also provides a method of screening candidateanti-cancer substances, using the Enigma-dependent expression level ofMdm2 or p53.

The present invention also provides a method of screening candidateanti-cancer substances, using an interaction level between Enigma andMdm2.

The present invention also provides a method of diagnosing cancers,identifying therapeutic results, or assessing prognosis, using an Enigmaexpression level in cancer cells.

The present invention also provides a kit in use for diagnosing cancers,identifying therapeutic results, or assessing prognosis, including aninhibitor of Enigma expression or activity.

The present invention also provides a use for utilizing an inhibitor ofEnigma expression or activity for preparation of an anti-cancercomposition.

The present invention also provides a use for utilizing an inhibitor ofEnigma expression or activity for preparation of an anti-canceradjuvant.

The present invention also provides a use for utilizing an inhibitor ofEnigma expression or activity for manufacturing a kit in use fordiagnosing cancers, identifying therapeutic results, or assessingprognosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is a schematic diagram which is indicative of illustrating avector including siEnigma;

FIG. 2 is a schematic diagram which is indicative of illustrating amethod of preparing adenovirus expressing F-Enigma;

FIG. 3 is a schematic diagram which is indicative of illustrating amethod of preparing adenovirus containing siEnigma;

FIG. 4A is a Western blot image illustrating through degradation pathwayof ubiquitin proteasome Mdm2 is stabilized and p53 is degraded whenEnigma is expressed;

FIG. 4B is a graph showing that a decrease in a p53 protein by Enigmaresults in a decrease in p53 activity by using a report analysis;

FIG. 4C is a Northern blot image illustrating the change in mRNA levelof Mdm2, p53, p21 in case of Enigma overexpression;

FIGS. 4D & 4E are Western blot images illustrating that Enigma regulatesMdm2 level p53-independently and regulates protein levels of p53 and p21regulation of p53 and p21 Mdm2-dependently;

FIGS. 5A & 5B Western blot images illustrating that an in-vivo bindingbetween Enigma and Mdm2 occurs p53-independently and Enigma-p53 bindingoccurs Mdm2-dependently;

FIG. 5C is a Western blot image illustrating that an in-vitro bindingbetween Enigma and Mdm2 occurs p53-independently and Enigma-p53 bindingoccurs Mdm2-dependently;

FIG. 5D is a schematic diagram and a Western blot image which indicatean Mdm2 domain (400-491 amino acids) which Enigma binds to;

FIGS. 5E & 5F are schematic diagrams and Western blot images whichindicate that LIM3 domain of Enigma which Mdm2 binds to;

FIG. 5G is a schematic diagram illustrating the Enigma-Mdm2-p53 ternarycomplex;

FIG. 6A is a Western blot image illustrating Enigma homolog (ENHk) doesnot bind to mdm2;

FIG. 6B is a Western blot image illustrating ENH has no effect onprotein levels of Mdm2 and p53 in cells;

FIGS. 7A & 7B are Western blot images illustrating when Enigma isoverexpressed in cells, Mdm2's self-ubiquitination is inhibited andp53's ubiquitination is increased; when Enigma expression is inhibited,the event is reversed;

FIG. 7C is a Western blot image illustrating a LIM3 domain of Enigmabinds to Mdm2 to inhibit Mdm2's self-ubiquitination;

FIG. 7D is a Western blot image illustrating Mdm2 increasinglyubiquitinates p53 by Enigma;

FIGS. 8A & 8B is to prove that Enigma has no effect on Mdmd2ubiquitination by PCAF both in vitro and in vivo;

FIG. 9A is a schematic diagram of Enigma promoter-reporter constructs inwhich SRE is present or absent in the promoter region of Enigma;

FIG. 9B is a diagram which indicates that an Enigma promoter isactivated by serum by using a report analysis;

FIGS. 9C & 9D are graphs showing that the SRE is essential for theserum-, FGF-, or HGF-mediated activation of Enigma promoter;

FIGS. 9E & 9F are the analytical results of EMSA and CHIP diagrams whichindicate that SRF binds specifically to an SRE portion on an Enigmapromoter;

FIGS. 10A & 10B are Western blot images and RT-PCR images illustratingthat serum increases Enigma expression following SRF induction attranscription level and thereby protein level of Mdm2 increases andprotein level of p53 is decreased;

FIGS. 10C & 10D are Western blot images illustrating Mdm2 stabilization,which is induced by HGF, is dependent on SRF and Enigma;

FIG. 10E is a Western blot image illustrating the effect of HGF on theSRF-Enigma-Mdm2 pathway is dependent on an MAP kinase;

FIGS. 10F to 10H are Western blot images and RT-PCR images illustratingthat the effect of HGF on the SRF-Enigma-Mdm2 pathway occurs in mice;

FIGS. 11A & 11B are Western blot images and RT-PCR images illustratingthe effect of serum removal on the SRF-Enigma-Mdm2 pathway;

FIGS. 12A & 12B are Western blot images and RT-PCR images illustratingthat the effect of serum on the SRF-Enigma-Mdm2 pathway occurs in humanfibroblasts in a manner dependent on an MAP kinase;

FIGS. 12C & 12D are Western blot images and RT-PCR images illustratingthat the effect of serum on the SRF-Enigma-Mdm2 pathway occurs in mouseembryonic fibroblasts in a manner dependent on an MAP kinase;

FIG. 13A is a Western blot image illustrating that SRF and Enigmaproteins are co-expressed with Mdm2 in human liver and stomach cancertissues;

FIG. 13B is an immunofluorescent staining of tumor tissues showing thatEnigma is co-localized with Mdm2 at apical cytoplasm of human stomachand colorectal tumor cells.

FIGS. 14A & 14B are a cell count method and a crystal violet stainingimage showing that Enigma weakens adriamycin (ADR)-mediated cytotoxiceffect p53-dependently and increase the cell viability;

FIG. 14C is a Western blot image illustrating the change in proteinlevels of Mdm2 and p53 according to the expression amount of Enigma inthe presence of ADR;

FIG. 14D is an image by a TUNEL method and a cell cycle analysisillustrating Enigma regulates cell viability p53-dependently in thepresence of ADR;

FIGS. 15A & 15B show Enigma's effect on proliferation of cell lineswhere p53 is unexpressed;

FIGS. 15C & 15D are Western blot images and crystal violet stainingimages investigating protein levels of Mdm2 and p53 and cell viabilityin the condition above;

FIGS. 16A & 16B are graphs by a cell counting method and Western blotimages showing that Enigma induces resistance to ADR in HLK3 hepatomacell line, and chemo-resistance is increased when Enigma expression isincreased;

FIGS. 16C & 16D are graphs by a cell counting method and Western blotimages that show that Enigma confers chemo-resistance by regulatingprotein levels of Mdm2 and p53 in the presence of etoposide or nutlin3a;

FIGS. 16E & 16F are a graph and a mass image showing that resistance toADR occurs when Enigma is overexpressed in mouse tumor model, and tumorgrowth is effectively inhibited when Enigma expression is inhibited;

FIG. 16G is a Western blot image illustrating the change in expressionof Enigam-Mdm2-p53 in tumors excised from mice;

FIG. 17A is a crystal violet staining image of an HLK cell in thecondition of FIG. 16A, and FIG. 17B illustrates an analysis result ofcycle of an HLK cell in the condition of FIG. 16A.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Features and advantages of the present invention will be more clearlyunderstood by the following detailed description of the presentpreferred embodiments by reference to the accompanying drawings. It isfirst noted that terms or words used herein should be construed asmeanings or concepts corresponding with the technical sprit of thepresent invention, based on the principle that the inventor canappropriately define the concepts of the terms to best describe his owninvention. Also, it should be understood that detailed descriptions ofwell-known functions and structures related to the present inventionwill be omitted so as not to unnecessarily obscure the important pointof the present invention.

The terms used herein are defined as below.

As used herein, the term “prevention” refers to all acts to inhibit thegrowth or transition of cancer by administering a composition of thepresent invention.

As used herein, the term “treatment” refers to all acts to improve orchange beneficially cancer symptoms by administering a composition ofthe present invention.

As used herein, the term “administering” refers to providing acomposition of the present invention for an individual by any suitablemethod.

As used herein, the term “individual” refers to all animals such ashuman, ape, dog, goat, pig or mouse, etc., with diseases for which acomposition of the present invention is administered to possibly improvecancer symptoms.

As used herein, the term “pharmaceutically effective amount” refers toan amount sufficient for treatment as a reasonable benefit or risk ratioapplicable to medical treatment, which may be determined from factorsincluding type of cancer, severity, medication activity, sensitivity tomedication, administering time, administering route and excretion ratio,treatment period, co-administering medication, and other well-knownfactors in the art.

The present invention is described in details as below.

The present invention provides a method of decreasing stability of Mdm2and of increasing p53 activity by inhibiting Enigma expression oractivity.

The present inventors identified that the stability of Mdm2 is regulateddependently on Enigma expression (See FIGS. 4A, 4D and 4E), and that,since Mdm2 is a E3 ligase with p53 as a substrate, the Mdm2stabilization results in enhancing ubiquitination and degradation ofp53, thereby ablating the antiproliferative activities of p53 (See FIGS.4A to 4E), and that Enigma inhibits the self-ubiquitination of Mdm2,resulting in stabilization of Mdm2, and stimulates Mdm2-mediatedubiquitination of p53 (See FIGS. 7A to 7D).

Furthermore, the present inventors observed the function of Enigma inthe cell proliferation inducing condition caused by growth promotingfactor, to investigate the effects on cell growth by Enigma's regulationof Mdm2-p53 expression. Resultantly, revealed was that Enigma expressionwas enhanced by SRF and Enigma plays a role in assisting cellproliferation by stabilizing Mdm2 and destabilizing p53 (See FIGS. 9A to9E and FIGS. 10A to 10E).

The inhibition of Enigma expression or activity of the present inventionis preferred, but is not limited, to use those selected from the groupconsisting of substances of inhibiting transcription of an Enigma gene,substances of inhibiting translation of transcribed Enigma mRNA, orsubstances of inhibiting the function of an Enigma protein.

The transcription inhibitor is preferred, but is not limited, to use apromoter, an enhancer, a protein or compound binding to a transcriptionregulating factor which binds to a promoter.

A substance of inhibiting the translation of mRNA is preferred, but isnot limited, to use a low-molecular-weight compound, RNA, siRNA or shRNAusing an antisense nucleic acid sequence or RNAi technique.

Details for them will be specifically described below:

1) RNAi

RNA interference (RNAi) is a post-transcriptional gene silencingmechanism where degradation of a corresponding mRNA occurs by inducing adouble-stranded RNA (dsRNA), which is corresponding to an Enigma gene,into a cell or organism. Since, by the RNAi effect, multiple celldivisions are maintained prior to a comeback of gene expression, RNAi isa very strong method of making a knockout or ‘knockdown’ which is aimedat the RNA level. It was observed that RNAi has been identified assuccessful in human cells including human embryonic kidney and HeLacells (Elbashir et al. Nature 411, 494-498, 2001).

Standard methods in molecular biology are used for RNAi technology ingene silencing. dsRNA, which corresponds to the sequence of a targetgene to be inactivated, may be produced by a standard method, forexample, a double-stranded simultaneous transcription of template DNAusing T7 RNA polymerase. A dsRNA production kit in use for RNAi mayinclude commercially available products (for example, a product made byNew England Biolabs, Inc.). Transfection methods of dsRNA and aprocessed plasmid for producing dsRNA are commonly known in the art.

2) siRNA

siRNA refers to a short, and double-stranded RNA which may induce RNAiphenomenon by a cleavage of a specific mRNA.

siRNA is not limited to a perfectly-paired RNA portion in which two RNAstrands pair up, and may include nonpairing portions due to mismatch(the corresponding bases are not complimentary), bulge (lacking in abase corresponding to one stand), etc. The length of a paired base is 10to 40 base pairs, preferably 15 to 30 base pairs to disturb Enigmaactivity and expression. The terminal structure of siRNA may be eitherblunt terminus or a terminal overhang if siRNA can silence the targetgene expression due to the RNAi effect. The cohesive terminal structuremay include a 5′ overhanging structure as well as a 3′ overhangingstructure. The number of overhanging bases is not limited. For example,the number may be 1 to 8 base pair(s), preferably 2 to 6 base pairs. Thelength of siRNA herein is that of paired polynucleotide. One end ofsiRNA may have a low-molecular-weight RNA (for example, a natural RNAmolecule such as tRNA, rRNA, viral RNA, or an artificial RNA molecule)if the inhibitory effect on a target gene expression can be maintained.The terminal structure of siRNA does not necessarily have the cut offstructure at both ends thereof, and may have a stem-loop structure inwhich an end of one strand of double-stranded RNA is connected by alinker RNA. There is no particular limitation in the length of thelinker unless that length hinders the paring of the stem portion.

Disclosure on a method of designing and producing siRNA with known genescan be referred to the following documents, for example, [Chalk A M,Wahlestedt C, Sonnhammer E L. Improved and automated prediction ofeffective siRNA Biochem. Biophys. Res. Commun. 2004 Jun. 18;319(1):264-74; Sioud M, Leirdal M., Potential design rules and enzymaticsynthesis of siRNAs, Methods Mol. Biol. 2004; 252:457-69]. Furthermore,to produce a modified and more stable siRNA, documents, e.g. [PCTLaid-Open Publication No. WO 2004/015107, U.S. Pat. Nos. 5,898,031 and6,107,094] may be referred.

A DNA-containing vector has been developed, which can produce siRNA incells. The method generally includes transcription of a short hairpinwhich is effectively processed to form siRNA in cells. The documentsrelated to such method may be referred to: [Paddison et. al., PNAS 2002,99:1443-1448; Paddison et. al., Genes & Dev 2002, 16:948-958; Sui et.al., PNAS 2002, 8:5515-5520; and Brummelkamp et. al., Science 2002,296:550-553]. The documents describe methods for producing siRNA whichspecifically targets various endogenously and exogenously expressedgenes. On delivery of siRNA, for example, the documents may be referredto: [Shen et. al., FEBS letters 539:111-114 (2003); Xia et. al., NatureBiotechnology 20:1006-1010 (2002); and Reigh et. al., Molecular Vision9:210-216 (2003)]. siRNA was successfully used for inhibition inprimate, and further detailed explanations may be referred to a document[Tolentino et. al., Retina 24(1) February 2004 pp 132-138].

On uses of siRNA as a therapeutic agent, for example, the followingdocuments may be referred to: [Korean Patent Registration No.10-0653738, Korean Patent Registration No. 10-0749859, Korean PatentRegistration No. 10-0930282, and Korean Patent Registration No.10-0810034].

3) Antisense Nucleic Acid Sequence

For nucleic acid encoding Enigma, antisense nucleic acid molecules maybe used as an inhibitor. ‘Antisense nucleic acid’ includes nucleic acidsequence complementary to a ‘sense nucleic acid’ encoding Enigma, forexample, complementary to a coding strand of a double-stranded cDNA orcomplementary to mRNA sequence. Thus, the antisense nucleic acid mayform hydrogen bonds with the sense nucleic acid. The antisense nucleicacid may be complementary to the entire coding strand or its part (e.g.a coding region). Although the antisense nucleic acid molecule may becomplementary to the entire coding region of an Enigma mRNA, antisenseoligonucleotide is more preferable for only a part (e.g. a translationinitiation portion) of the coding or non-coding region of the EnigmamRNA. Antisense oligonucleotide may be, for example, about 5 to 50 ntlong. Antisense nucleic acid may be constructed by using chemicalsynthesis and enzyme linked reaction according to well-known methods.For example, it may be very easy to produce antisense nucleic acid usingchemical synthesis method such as phosphoramidite chemistry ofsulfurizing acetonitrile into tetraethylthiuram disulphide, which isdisclosed in the document [Tetrahedron Lett. 32, 30005-30008, 1991].Examples of a modified nucleotide in use for production of the antisensenucleic acid may be 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil, 1-methylinosine, 2,2-dimethylguanine,2-methyladenine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 5-carboxymethylaminomethyl-2-thiouridine,3-(3-amino-3-N-2-carboxypropyl)uracil, 5′-methoxycarboxymethyluracil,5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, 1-methylguanine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 2,6-diaminopurine,5-methyl-2-thiouracil, pseudouracil, queosine, 2-thiocytosine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,5-methyl-2-thiouracil, (acp3)w and wybutoxosine. As occasion arises, theantisense nucleic acid may be biologically generated by using expressionvectors.

Although it is preferable that substances inhibiting function of theEnigma protein may include peptide binding to the protein, antibody,compound, peptide mimetics, etc., the present invention is not limitedthereto.

Details for them will be specifically described below:

1) Peptide Mimetics

It is possible to inhibit an original Enigma polypeptide from binding toMdm2 by producing mimetics (e.g. peptide or non-peptidepharmaceuticals), which inhibits a protein-binding domain of Enigmapolypeptide (EU Patent Application EP 0412765 and EP 0031080).

Main residues of a non-hydrolyzed peptide analog may be generated byusing 13-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647,1985), keto-methylene pseudopeptide analogs (Ewenson et al. J Med Chem29:295, 1986; and Ewenson et al. in Peptides: Structure andFunction(Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), azepine (Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), benzodiazepine (Freidinger et al. in Peptides;Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), β-amino alcohol (Gordon et al. Biochem Biophys ResCommun 126:419 1985), and substituted gamma lactam ring (Garvey et al.in Peptides: Chemistry and Biology, G. R. Marshell ed., ESCOM Publisher:Leiden, Netherlands, 1988).

The present invention provides a method of producing Enigma-siRNAexpression vector (siEnigma) to inhibit Enigma expression.

In specific, based on a method disclosed in Korean Patent RegistrationNo. 877824 (U. S. patent application Ser. No. 12/093093(May 08, 2008)),a recombinant vector was produced as it is complementary to mRNA ofEnigma, preferably is indicated as SEQ ID NO: 2 or SEQ ID NO: 5(5′-AAAGACCTTCTACTCCAAGAA-3′ or 5′-AATGCCATGGCTGTGACTTCA-3′,respectively), and cloned into a pSuper plasmid vector to be expressedby an H1 promoter. Also, an Enigma-siRNA expression vector foradenovirus production was prepared, which contains an H1 promoter,Enigma-siRNA, and even five T bases (T₅) of transcription terminationsequences, by processing the pSuper plasmid vector with a restrictionenzyme into a pShuttle vector for adenovirus production that allowsgenes to be transferred and expressed into a cell. Additionally, afterEnigma was cloned into a pCMV-Taq2b vector such that the mRNA of Enigmawas expressed by a CMV promoter, and thereafter adenovirus F-Enigma wasproduced to allows Enigma to be transferred into a cell (see FIGS. 1 to3, and U. S. patent application ser. No. (May 08, 2008) 12/093,093;Korean Patent Registration No. 877824).

Also, the present invention provides an anti-cancer composition or ananti-cancer adjuvant, which includes an Enigma expression inhibitor oran Enigma activity inhibitor as an effective ingredient.

The Enigma expression inhibitor may be selected from the groupconsisting of antisense oligonucleotide complimentarily binding to mRNAof an Enigma gene, short interfering RNA, short hairpin RNA, and RNAi,however, the present invention is not limited thereto.

The Enigma activity inhibitor may be selected from the group consistingof compound complimentarily binding to an Enigma protein, peptide,peptide mimetics, and antibody, however, the present invention is notlimited thereto.

The cancer may be selected from the group consisting of stomach cancer,liver cancer, and colon cancer, however, the present invention is notlimited thereto.

The present inventors found out that Enigma is co-expressed with Mdm2 invarious type of cancer tissues (see FIGS. 13A and 13B), that Enigmaoverexpression in cancer cells induces resistance to anti-cancer agents,and that the treatment of siEnigma inhibiting Enigma expression inducesapoptosis of cancer cells effectively (see FIGS. 14A to 14D; FIGS. 16Ato 16G; and FIG. 17). Thus, an Enigma expression inhibitor or an Enigmaactivity inhibitor may be usefully utilized as an effective ingredientfor anti-cancer therapeutic agent by efficiently inducing apoptosis ofcancer cells, and may be usefully utilized as an effective ingredientfor anti-cancer adjuvant by inhibiting resistance to anti-cancer agents.

The anti-cancer composition or anti-cancer adjuvant of the presentinvention includes 0.0001 to 50% by weight of the effective ingredientrelative to the total weight of the composition.

The anti-cancer composition or anti-cancer adjuvant of the presentinvention may include one or more type of other effective ingredient(s)showing an identical or similar function in addition to the effectiveingredient.

The anti-cancer composition or anti-cancer adjuvant of the present maybe produced containing one or more type of a pharmaceutically acceptablecarrier(s) in addition to the effective ingredient described above. Asthe pharmaceutically acceptable carrier, saline solution, sterilizedwater, linger's solution, buffer saline, dextrose solution, maltodextrinsolution, glycerol, ethanol, liposome, and at least one combinationthereof, may be used, and if necessary, other typical additives such asantioxidants, buffer solution, bacteriostatic agents, etc., may beadded. Moreover, it can be formulated in the form of an injectableformulation such as aqueous solution, suspension and emulsion, a pill, acapsule, a granule, or a tablet by supplementarily adding diluent,dispersing agent, surfactant, binder and lubricant. And it may be usedcombining a target-specific antibody or other ligands with the carrierto act specifically upon a target organ. Furthermore, it is possible topreferably formulize according to each disease or ingredients using asuitable method in the art, for example, a method disclosed inRemington's Pharmaceutical Science (the latest edition), Mack PublishingCompany, Easton Pa.).

Further, the present invention provides a method of treating cancer,including administrating an Enigma expression inhibitor or an Enigmaactivity inhibitor in a pharmaceutically effective amount to anindividual suffering from cancer.

The Enigma expression inhibitor may be selected from the groupconsisting of antisense oligonucleotide complimentarily binding to mRNAof an Enigma gene, short interfering RNA, short hairpin RNA, and RNAi,however, the present invention is not limited thereto.

The Enigma expression inhibitor may be selected from the groupconsisting of compound complimentarily binding to an Enigma protein,peptide, peptide mimetics, and antibody, however, the present inventionis not limited thereto.

The cancer may be selected from the group consisting of stomach cancer,liver cancer, and colon cancer, however, the present invention is notlimited thereto.

The administering method is not particularly limited, and thus it may bea parenteral administering (for example, applied intravenously,subcutaneously, intraperitoneally, or locally) or oral administering.Although it is preferable to administer parenterally, more preferable toadminister subcutaneously, the present invention is not limited thereto.

The range of dosage varies according to a patient's body weight, age,sex, health status, diet, dose time, dose method, excretion rate, theseverity of disease, etc. The daily dosage for a compound is in therange of about 0.1 to 100 mg/kg, preferably 0.5 to 10 mg/kg. It ispreferable to administer the formulation one or more times a day,however, the present invention is not limited thereto.

The present inventors observed that Enigma is co-expressed with Mdm2 invarious types of cancer tissues, that Enigma overexpression in cancercells induces resistance to anti-cancer agents, and that the treatmentof siEnigma inhibiting Enigma expression induces effective apoptosis ofcancer cells. Consequently, the present inventors found out that anEnigma expression inhibitor or an Enigma activity inhibitor may be usedas an effective ingredient for anti-cancer therapeutic agent by inducingeffective apoptosis of cancer cells.

Also, the present invention provides a method of screening candidatesubstances for anti-cancer agents, which uses an Enigma-dependentexpression level of Mdm2 or p53. In the present invention, since Mdm2expression is increased due to its stabilization depending on Enigmaexpression and P53 expression is decreased via the Mdm2, it is possibleto screen a substance having anti-cancer activity by selecting asubstance that allows the expression of the Enigma or Mdm2 to bedecreased or the expression level of p53 to be increased.

In particular, the screening method may include:

1) treating candidate substances on cells expressing Enigma and Mdm2;

2) measuring a binding level for Enigma and Mdm2; and

3) selecting candidate substances to decrease the binding level forEnigma and Mdm2, compared to a control group untreated with candidatesubstances. However, the present invention is not limited to the above.

Furthermore, the screening method may include:

1) treating candidate substances on cells expressing Enigma, Mdm2 andp53;

2) measuring a binding level for Enigma and Mdm2; and

3) selecting candidate substances to decrease the binding level forEnigma and Mdm2, compared to a control group untreated with candidatesubstances. However, the present invention is not limited to the above.

A candidate substance as set forth in the screening method may beselected from the group consisting of a nucleic acid, a protein, otherextracts, a compound and a natural substance, however, the presentinvention is not limited to the above.

An expression level as set forth in the screening method may be measuredby measuring the transcriptional activity level of genes or by measuringthe amount of expressed proteins, however, the present invention is notlimited to the above.

The transcriptional activity level may be measured through LuciferaseAssay and the amount of proteins may be measured through Western blot,however, the present invention is not limited to the above.

Furthermore, the present invention provides a method of screening ananti-cancer composition, using a binding level for Enigma and Mdm2.

The present inventors identified that Enigma and Mdm2 interactsdirectly, and Enigma and p53 interacts indirectly via Mdm2 (see FIGS. 5Ato 5C). Also, the present inventors identified that the LIM3 portion ofEnigma interacts with 401 to 491 (a RING domain portion) of Mdm2 (seeFIGS. 5D to 5G). Thus, it can be understood that substance inhibiting aninteraction between Enigma and Mdm2 may become anti-cancer active byweakening the stabilization of Mdm2 and activating p53.

The screening method may include:

1) contacting Enigma and Mdm2 in the presence or absence of candidatesubstances;

2) measuring a binding level for Enigma and Mdm2; and

3) selecting candidate substances to decrease the binding level forEnigma and Mdm2, compared to the absence of candidate substances.However, the present invention is not limited to the above.

Furthermore, the screening method may include:

1) bringing cells expressing Enigma and Mdm2 into contact with candidatesubstances;

2) measuring a binding level for Enigma and Mdm2; and

3) selecting candidate substances to decrease the binding level forEnigma and Mdm2, compared to a control group untreated with candidatesubstances. However, the present invention is not limited to the above.

A candidate substance as set forth in the screening method may beselected from the group consisting of a nucleic acid, a protein, otherextracts, a compound and a natural substance, however, the presentinvention is not limited to the above.

Binding as set forth in the screening method may be measured by animmunoprecipitation method. Immunoprecipitation, for example, may beperformed by a method in a document (Harlow and Lane, Antibodies,511-52, Cold Spring Harbor Laboratory publications, New York, 1988).SDS-PAGE is generally used for analysis of immunoprecipitated proteins,and binding proteins may be analyzed by the molecular weight of proteinsusing gel of a suitable concentration.

As set forth in the screening method, a two-hybrid system, which usescells, may be employed (“MATCHMAKER Two-Hybrid system”, “MATCHMAKERMammalian Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system”(Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); Reference:Dalton and Treisman, Cell 68: 597-612, 1992; Fields and Stemglanz,Trends Genet. 10: 286-92, 1994).

In the screening method, a biosensor using surface plasmon resonancephenomenon may be used as a means of detecting or quantifying substancesbound in the present invention. In use of the biosensor, interaction bythe binding may be observed real-time as a surface plasmon resonancesignal.

The present invention provides a method of diagnosing cancer,identifying treatment results, or assessing prognosis, the methodincluding measuring an Enigma expression level in cancer cells, usingone or more of antibody reactive with Enigma and nucleic acidcomplementary to Enigma genes.

Furthermore, the present invention provides a kit in use for diagnosingcancers, including one or more of antibody reactive with Enigma andnucleic acid complementary to Enigma genes.

The present inventors observed Enigma overexpression in cancer cells,and resultantly confirmed that SRF and Enigma were co-expressed withMdm2 in human liver and stomach cancer cells (see FIGS. 13A to 13B).Also, the present inventors found out that cancer cells' growth andresistance to anti-cancer agents were induced dependently on p53 and anEnigma expression level in cancer cells (see FIGS. 14A to 14D; FIGS. 16Ato 16G; FIG. 17). Therefore, measuring the level of Enigma expressionmake it possible to know the extent of apoptosis of medicaments relativeto cancer cells in cancer therapy, and thus may be used as a method ofassessing the prognosis of anti-cancer therapy.

In the method of diagnosing cancers, detection of Enigma expressionlevel higher than a normal range tells that a patient is suffering fromcancer. Furthermore, in a diagnosis reagent of an individual who hasundergone or is undergoing cancer therapy, detection of Enigmaexpression level in the normal range tells a success of cancer therapy,and detection of Enigma expression higher than the normal range in thediagnosis reagent tells that the cancer therapy should continue to beapplied. Furthermore, in a diagnosis reagent of an individual who issuffering from cancer, detection of normal Enigma expression level inthe normal range tells the prognosis is good, however, detection ofEnigma expression level higher than the normal range in the diagnosisreagent tells that the prognosis is bad.

A kit of the present invention in use for diagnosing cancers may includeone or more substance, which is reactive to Enigma, and a reagent fordetecting reaction product and instructions related thereto. Forexample, one or more substance which is reactive to Enigma may be an RNAor DNA complementary to RNA or DNA of Enigma, and an antibody whichbinds to an Enigma protein. A reagent for detecting reaction product maybe a nucleic acid, a protein marker and a color reagent.

The present invention also provides a use for utilizing an Enigmaexpression inhibitor or an Enigma activity inhibitor for preparation ofan anti-cancer composition.

The present invention also provides a use for utilizing an Enigmaexpression inhibitor or an Enigma activity inhibitor for preparation ofan anti-cancer adjuvant.

The present invention also provides a use for utilizing an Enigmaexpression inhibitor or an Enigma activity inhibitor for manufacturing akit in use for diagnosing cancers, identifying therapeutic results, orassessing prognosis.

The Enigma expression inhibitor may be selected from the groupconsisting of antisense oligonucleotide complimentarily binding to mRNAof an Enigma gene, short interfering RNA, short hairpin RNA, and RNAi,however, the present invention is not limited thereto.

The Enigma activity inhibitor may be selected from the group consistingof substance complimentarily binding to an Enigma protein, peptide,peptide mimetics, antibody, and compound, however, the present inventionis not limited thereto.

The cancer may be selected from the group consisting of stomach cancer,liver cancer, and colon cancer, however, the present invention is notlimited thereto.

The present inventors observed that Enigma is co-expressed with Mdm2 invarious types of cancer tissues, that Enigma overexpression in cancercells induces resistance to anti-cancer agents, and that the treatmentof siEnigma inhibiting Enigma expression induces effective apoptosis ofcancer cells. Consequently, the present inventors found out that anEnigma expression inhibitor or an Enigma activity inhibitor may be usedas an effective ingredient for anti-cancer therapeutic agent by inducingeffective apoptosis of cancer cells, and may be used as an effectiveingredient for anti-cancer adjuvant by inhibiting resistance toanti-cancer agents.

Herebelow examples of the present invention will be described indetails.

The examples below exemplify only the present invention, and thus itshould be construed that the present invention is not limited to theexamples below.

EXAMPLE 1 MDM2 Stabilization by Enigma and p53 Degradation Thereby

The present inventors cloned fragments obtained by a common PCR methodinto pCMV Taq2B (Stratagene, USA) to thereby produce a vector whichoverexpresses Enigma, by using a vector containing Enigma cDNA (SEQ IDNO: 1), which was furnished by Korea Research Institute of Bioscienceand Biotechnology (KRIBB) as a template. Furthermore, the presentinventors cloned a nucleotide fragment of 5′-AAAGACCTTCTACTCCAAGAA-3′SEQ ID NO: 2) that inhibits Enigma expression specifically into on theHind III/Bgl II sites of pSuper plasmid vector (FIG. 2). Also, for easyintroduction into a cell, adenovirus, containing siEnigma (FIG. 3), andadenovirus containing Flag-Enigma (FIG. 2) were produced in followingmanner, based on the method described in Korean Patent Registration No.627377 (Hepatology 43, 1042-1052, 2006) and 877824 (U. S. patentapplication Ser. No. 12/093,093(May 08, 2008); Nat Med 12, 809-816,2006).

The pSuper Enigma-siRNA plasmid prepared in advance was processed intopShuttle (BD Bioscience, USA) vector for adenovirus production withVbaI/HindIII, and then a DNA fragment containing T₅ transcriptionaltermination sequence was cloned at H1 promoter (pShuttle/Enigma-siRNA).To prepare adenovirus expressing F-Enigma, only an Enigma portion wascut from the prepared pCMV taq2B-Enigma with NotI/XhoI and by thencloned into a pCMV shuttle vector. A recombination of pAdEasy-1, whichincludes adenovirus genome, with pShuttle/Enigma-siRNA andpCMVshuttle-F-Enigma was performed by being simultaneously transduced toE. coli strain BJ5183. A plasmid containing recombinant adenovirusgenome, which was obtained by homologous recombination in the E. coli,was identified through restriction enzyme analysis after separating DNAfrom an E. coli colony grown in Kanamycin selective medium.

Production of adenovirus particles in a plasmid containing recombinantadenovirus genome was performed as below. A plasmid containingrecombinant adenovirus genome was cut with PacI so that terminal repeat(TR), a replication origin of adenovirus, is located at both ends oflinearized DNA, and was transfected into an HEK293 cell (ATCC), whichwas grown in 60-mm culture plate by 70-80% confluency, using a calciumphosphate method. A plasmid containing recombinant adenovirus genome wasintroduced. Cells which generate adenovirus particles became cytopathicafter 2-3 days, and formed plaques after 4-5 days. Cells which becamecytopathic were collected and were froze-and-thawed, thereby releasingadenoviruses from the cells. Supernatants of the cell lysates wereobtained by centrifugation. Cells, which were prepared in a 100-mmculture plate by 60-70% confluency, were infected with the supernatantof the cell lysates. Once cells die by proliferation after infection ofadenovirus, the cells were recollected and then repeated the aboveprocedure to create correct recombinant adenovirus. The number ofviruses was determined by the plaque-forming method.

<1-1> Enigma Stabilizes Mdm2

Enigma's effect on ubiquitin-proteasome-dependent proteolysis of Mdm2was identified by the following method: A Flag-Enigma expression vectorwas induced into an HLK3 cell line (Chonbuk National University MedicalSchool, South Korea) concentration-wise (+: 2 μg, ++: 5 μg). Proteinlevels of Enigma and Mdm2 were determined by Western blotting aftercollecting cells in the presence or absence of MG132 (10 μM) for 12hours. As a result, while Mdm2 protein level was elevated dependently onEnigma expression, protein levels of p53 and p21 decreased (FIG. 4A).

<1-2> p53 Protein Level Reflects p53 Activity

To identify whether a decrease in p53 protein level by Enigma leads to adecrease in p53 activity, activity of target gene promoters regulated byp53 was examined. After reporter genes (0.5 μg) such as p53RE-Luc,Bax-Luc, p21-Luc were introduced into HLK3 cell line, or p53^(+/+) orp53^(−/−) colon cancer cell line (HCT116, ATCC) concentration-wise withFlag-Enigma (+: 2 μg, ++: 5 μg) respectively, and the cells were treatedor not treated with MG132 (10 μM) for 12 hours, a reporter assay(Promega, USA) on collected cells was performed. As the result,transcriptional activity of target genes regulated by p53 was shown tobe inhibited concentration-dependently on Enigma expression (FIG. 4B).

<1-3> Enigma's Effect on mRNA Levels of Mdm2, p53, and p21

A Northern blotting was performed to test whether an expression changein Mdmd2 and p53 by Enigma occurred at the level of mRNA. A Flag-Enigmaexpression vector was transfected into an HLK3 cell line at twodifferent doses (+: 2 μg, ++5 μg). Total RNA was extracted with RNeasykit (Qiagen) after 48 hours, and a Northern blotting was performed witheach probe (P1/P2 Mdm2, p53, p21). As the result, the amount of mRNA inP1-Mdm2, p53, and p21 was unvaried irrespective of the amount of Enigmaexpression, and the amount of mRNA in P2-Mdm2, which is regulated byp53, decreased dependently on Enigma expression (FIG. 4C). The resultsuggests that Enigma regulates Mdm2 and p53 levels at thepost-transcriptional step.

<1-4> Enigma regulates p53 level Mdm2-dependently Mdm2^(−/−)p53^(−/−)MEF and Mdm2^(+/+)p53^(−/−) MEF cell lines (G. Lozano, M. D. ANDERSONCANCER CENTER) were used to identify whether an expression inhibition ofp53 and p21 by Enigma is Mdm2-dependent. At 16 hours after an adenovirusvector (Ad-p53) was infected into each of said cell lines, Ad-F-Enimga(50 MOI), Ad-LacZ (50 MOI), Ad-siEnigma (100 MOI), Ad-siControl (100MOI) were infected again. After 32 hours, the cells were collected, anda change in expression for each protein was identified by a Westernblotting. As the result, expression of p53 and p21 decreased dependentlyon Mdm2 expression (FIG. 4D).

<1-5> Enigma Regulates p21 Level p53-Dependently

p53^(+/+), p53^(−/−) cell lines were used to identify whether expressioninhibition of p21 by Enigma is by way of p53 or not. Flag-Enigma,siEnigma were introduced to the p53^(+/+), p53 cell lines. After 48hours, protein levels of p53 and p21 were identified by a Westernblotting. As the result, a change in p21 is caused by a change in p53,and a direct effect by Enigma was not shown (FIG. 4E).

EXAMPLE 2 Identification of an Enigma-Mdm2-p53 Ternary Complex

The present inventors, to test interaction between Enigma and Mdm2,generated mutants (F-PDZ, F-PDZ-LIM1, F-LIM) of Enigma in pCMVtaq2Bvector by a common PCR cloning method, and produced mutants (GST-1-100,GST-101-491, GST-201-491, GST-301-491) of Mdm2 in pEBG vector by a PCRcloning method. His-Mdm2, Enigma, GST-p53, EniΔLIM3 proteins wererespectively separated and purified from E. coli to identify theinteraction in vitro.

<2-1> Enigma Interacts with p53 Through Mdm2 in Cells

Interaction between Enigma and Mdm2-p53 was identified by an IP methodin a colon cancer cell line (HCT116) of p53^(+/+) or p53^(−/−) andMdm2^(−/−)p53^(−/−) MEF and Mdm2^(+/+) MEF cell lines. As the result,interaction between Enigma and Mdm2 occurred irrespectively of p53, andin the presence of Mdm2, an Enigma-Mdm2-p53 complex was found to beformed (FIGS. 5A and 5B).

<2-2> Enigma Interacts with p53 Through Mdm2 In Vitro

In vitro binding assay was performed to identify directly in-vitrointeraction between respective proteins (1 μg His-Mdm2, 0.5 μg Enigma, 1μg GST-p53) which are separated and purified from E. coli. Bindingbetween respective proteins were induced after mixing, the bindingsbetween proteins were identified by a GST pull-down and an IP method. Asthe result, Enigma bound to Mdm2 directly, and bound to p53 when Mdm2was present (FIG. 5C).

<2-3> Determination of a Binding Site Between Enigma and Mdm2

A binding site was determined by using respective mutants produced fromEnigma and Mdm2. First, each mutant vector (5 μg) was introduced into a293 cell line where Hag-Enigma is constitutively expressed, andafterwards binding was identified by GST pull-down and by a Westernblotting with a Hag antibody (FIG. 5D). Also, each Enigma mutant (5 μg)and GST-Mdm2 expression vector (5 μg) were introduced into a 293T cellline (ATCC) by a calcium phosphate method. At 24 hours, the cells werecollected, and binding was identified by IP with a Flag antibody and bya Western blotting with a GST antibody (FIG. 5E). Bacterially expressedEniΔLIM3, in which the LIM3 domain of Enigma was removed, were not boundto Mdm2 (FIG. 5C), and Mdm2 (401-491) and LIM3 domain of Enigma wereshown as binding site. By identifying interaction between a GST-LIM3mutant, which includes a LIM3 site only, and Mdm2 after an induction ofcell lines by GST pull-down and by a Western blotting with an Mdm2antibody, the present inventors could identify that the LIM3 site ofEnigma binds to 401-491 site of Mdm2 (FIG. 5F). Thus, based on knownMdm2-p53 interaction, shown is that a “C” terminus of Enigma and a “C”terminus of Mdm2 are bound directly, with the binding structure of a “N”terminus of Mdm2 and a “N” terminus of p53 revealed (FIG. 5G).

<2-4> Enigma binds to Mdm2 Specifically

A binding between ENH (Enigma homolog), which retains a molecularstructure similar to Enigma, and Mdm2 was tested. Expression vectorssuch as Flag-ENH, Flag-Enigma and GST-Mdm2 were transfected into a 293Tcell line by a calcium phosphate method. After 48 hours when the cellswere collected, GST pull-down and a Western blotting with a Flagantibody were performed. As the result, no binding between Mdm2 and ENHoccurred (FIG. 6A). Furthermore, after an F-ENH expression vector wastransfected into an HLK3 cell line concentration-wise (2, 5 μg), noexpression change in Mdm2 and p53 was identified. As the result, ENH hadno effect on the amount of expression in Mdm2 and p53 (FIG. 6B). Thus,the binding between Enigma and Mdm2 and the increase in Mdm2 expressionthereby could be identified as specific interaction.

EXAMPLE 3 Enigma's Effect on Self-Ubiquitination of Mdm2 andUbiquitination of p53

The present inventors tested Enigma's effect on self-ubiquitination ofMdm2 and ubiquitination of p53 in vivo and in vitro to elucidate amechanism where Enigma binds directly to Mdm2 to stabilize Mdm2, andMdm2 enhances degradation of p53.

<3-1> Enigma's In Vivo Effect on Ubiquitination of Mdm2 and p53

Flag-Enigma (5, 10 μg), siEnigma (10μ, 15 μg) were induced into an HLK3cell line concentration-wise and treated with MG132 12 hours beforetheir collection. His-Ub (5 μg) and F-Enigma (5, 10 μg) were inducedinto another 293T cell line concentration-wise and treated with MG132 12hours before their collection. Cell protein extract was prepared with alysis solution, and IP was performed with respective antibodies; afluctuation in Mdm2-Ub and p53-Ub conjugates was identified by a Westernblotting with Ub and H is antibodies. As the result, the moreoverexpressed Enigma was in a cell, the more decreased Mdm2ubiquitination was, the more increased p53 ubiquitination was; the moreinhibited Enigma expression was, the more increased Mdm2 ubiquitinationwas, the more decreased p53 ubiquitination was (FIGS. 7A and 7B).

<3-2> Enigma Inhibits Self-Ubiquitination of Mdm2 and PromotesUbiquitination of p53

In vitro ubiquitination assay was performed to test self-ubiquitinationof Mdm2 by Enigma's binding. After ubiquitin reaction mixture (includingE1, E2, His-Ub, ATP) was added and reacted to His-Mdm2 protein (0.5 μg),His-Mdm2 (C464A: 0.5 μg), Enigma protein (0.5, 1 μg), F-EniΔLIM3 (1 μg),a change in self-ubiquitination and binding proteins for Mdm2 wereidentified by an IP/IB method. As the result, self-ubiquitination ofMdm2 was inhibited by F-Enigma, which binds to Mdm2, and Enigma had noeffect on an Mdm2 (C464A) variant where 464-cysteine on the Mdm2 RINGfinger region was substituted by alanine (FIG. 7C). Also, Enigmastimulated Mdm2-mediated p53 ubiquitination in vitro (FIG. 7D). Thus,Enigma was identified as a determining factor that shifts specificity ofMdm2 E3 ligase from itself toward p53.

<3-3> Enigma's Effect on PCAF-Mediated Mdm2 Ubiquitination

PCAF (p300-CBP-associated factor) induces the ubiquitination of Mdm2(Nat Cell Biol 9, 331-338, 2007). When proteins purified from bacteriaand described in FIG. 5-1 a were used to perform an in-vitro ubiquitinexperiment, Enigma had no effect on Mdm2 ubiquitination by PCAF (FIG.8A). This event was also tested in an Mdm2^(−/−)p53^(−/−) MEF cell line(FIG. 8B). The results suggest that Enigma inhibits specifically theself-ubiquitination of Mdm2 and enhances Mdm2-mediated ubiquitination ofp53.

EXAMPLE 4 Identification of Factors that Activate an Enigma Promoter

Since p53 has a function to inhibit cell proliferation and Enigmainduces Mdm2 stabilization in cells and causes protein level of p53 todecrease, whether an Enigma promoter is activated in a condition of cellproliferation and how the mechanism operates were investigated.

<4-1> Serum Activates an Enigma Promoter

The present inventors discovered that a serum response element (SRE)where a serum response factor (SRF) binds occurs on an Enigma promoter,and produced Enigma-Luc (pGL2), SRE-Luc, ΔSRE-Luc (pGL3) reportervectors respectively, by inserting a fragment, which includes or doesnot include SRE in a promoter sequence, into a pGL2/3 vector with aluciferase (FIG. 9A). After Enigma-Luc was induced into an HLK3 cellline (0.5 μg), was cultured in DMEM with 0.1% PBS for 24 hours, and wascultured with 5% and 20% FBS added for 6 hours again, luciferaseactivity of collected cells were measured. As the result, activity ofEnigma-Luc increased dependently on the concentration of serum (FIG.9B).

<4-2> Growth Factors Activate Enigma Promoter through SRE

After SRE-Luc (0.5 μg), ΔSRE-Luc (0.5 μg), which were produced to testSRF dependence of Enigma expression, were induced into an HLK3 cellline, the cells were cultured in DMEM with 0.1% FBS for 24 hours, andwere cultured with 5% and 20% FBS, FGF (20, 40 ng/ml), or HGF (20, 40ng/ml) added for 6 hours again, luciferase activity of collected cellswere measured. As the result, the reporter activity of SRE-Luc but notΔSRE-Luc increased dependently on the concentration of growth factor(FIGS. 9C and 9D).

<4-3> SRF Binds to an SRE Site of an Enigma Promoter

EMSA and in vivo ChIP analyses were performed to test whether SRF bindsspecifically to an SRE site [5′-CTATATAAGG-3′(SEQ ID NO: 3)] of anEnigma promoter. The present inventors, to perform the EMSA analysis,produced a nucleoprotein extract of HLK3, which was treated or nottreated with serum, brought the extract into reaction with an³²P-radiolabeled SRE nucleotide (5′-CTATATAAGG (SEQ ID NO: 3) X3), andthen brought the reaction mixtures into reaction with an anti-SRFantibody, and identified the binding in a 6% acrylamide gel. As theresult, revealed is that SRF binds specifically to the SRE site ofEnigma promoter (FIG. 9E).

Furthermore, the present inventors, for ChIP analysis performed toidentify whether SRF binds to an Enigma promoter in cells, fixed HLK3,which was treated or not treated with serum, with 1% formalin, and lysedcollected cells by sonic treatment. The present inventors identified byPCR with a particular primer whether SRF binds to an SRE site of anEnigma promoter after obtaining an SRF-DNA complex going through IP withan anti-SRF antibody. As the result, revealed is that SRF binds to anSRE site of an Enigma promoter in cells (FIG. 9F). The results abovesuggest that in a condition of cell proliferation an Enigma promoter isactivated by SRF and thereby the expression increases.

EXAMPLE 5 Serum or HGF Regulates SRF-Enigma-Mdm2-p53 Pathway

In a condition of cell proliferation or enhancing survival, an increasein Mdm2 has been reported (Growth factors 23, 183-192, 2005). Thepresent inventors tested whether Enigma expression by SRF wouldstabilise Mdm2, and inhibit p53 in a cell and a mouse liver to identifyif said event is caused by Enigma.

<5-1> Serum Regulates SRF-Enigma-Mdm2-p53 Pathway

The present inventors identified a change in protein by a Westernblotting and a change in transcriptional level (mRNA) by RT-PCR, whichuses total RNA separated condition-wise, respectively after culturing ap53^(+/+) or p53^(−/−) colon cancer cell line in a condition of removingserum, and again culturing the cell line in media containing 10% serumtime-wise. As the result, revealed is that SRF increased from thetranscriptional step, and therefore an amount of protein also increased,and an amount of protein in Enigma, which is transcriptionallyactivated, also increased. The increase in Enigma induced stabilizationof Mdm2 protein and decreased protein level of p53. A series of increasein SRF-Enigma-Mdm2 was independent from p53 (FIGS. 10A and 10B).

After culturing a p53^(+/+) or p53^(−/−) colon cancer cell line in mediacontaining 10% serum, the present inventors identified, by a Westernblotting and RT-PCR, changes in each molecule in a condition of removingserum time-wise. As the result, in a condition of restricted growth,mRNA and protein levels of SRF and Enigma are all decreased, proteinlevel of Mdm2 decreased, and protein level of p53 increased (FIGS. 11Aand 11B).

<5-2> HGF Regulates SRF-Enigma-Mdm2-p53 Pathway

The present inventors investigated whether SRF-Enigma-Mdm2 pathway isalso regulated by HGF. The present inventors, to show specific relevanceof SRF, Enigma, identified changes in each protein by a Western blottingafter inducing 10 μg each of siEnigma, siSRF into an HLK3 cell line,culturing them in a condition of removing serum, and processing eachwith HGF (40, 60 ng/ml) 4 hours before cell collection. As the result,an increase in SRF-Enigma-Mdm2 and a SRF- or Enigma-dependent decreasein p53 were identified (FIG. 10C). Furthermore, the present inventorsobserved a change in Enigma and Mdm2 after directly inducing F-SRF intoan HLK3 cell line concentration-wise (5, 10 μg). As the result, revealedis that SRF induced Enigma gene expression directly (FIG. 10D).

<5-3> An MAP Kinase Regulates SRF-Enigma-Mdm2 Pathway

Since an MAP kinase is known to be associated with SRF expression bycell growth factor (J Physiol Pharmacol 53, 147-157, 53, 2002), thepresent inventors hypothesized that an MAP kinase is associated withregulation of SRF-Enigma-Mdm2 pathway, and tested by using PD98059, MAPkinase-specific inhibitor. The present inventors treated the cells withHGF and with or without PD98059 after inducing Ad-p53 or Ad-LacZ intoMdm2^(+/+)p53^(−/−) MEF cell lines. As the result, PD98059 abolished theHGF effect on SRF-Enigma-Mdm2 pathway (FIG. 10E).

The present inventors identified serum response by using MRC5 (humannormal fibroblast), MEF (mouse embryonic fibroblast) to identifypresence of SRF-Enigma-Mdm2 pathway in normal cells. In case of MRC5,activity of SRF-Enigma-Mdm2 pathway by serum was inhibited when siSRF(10, 15 μg) is induced or PD98059 is present; in case of MEF, activityof SRF-Enigma-Mdm2 pathway was inhibited by PD98059 (FIGS. 12A and 12C).Thus, an amount of protein by transcriptional activity increased forSRF, Enigma, transcript level for Mdm2 (p1-Mdm2) and p53 was unvaried.However, transcript level for p2-Mdm2 changed dependently on an amountof p53 expression (FIGS. 12B and 12D). The results suggest thattranscriptome of SRF-Enigma is primarily, orderly generated when a cellproliferates, and there is a pathway where Enigma protein stabilize Mdm2and decrease p53.

<5-4> In Vivo Presence of SRF-Enigma-Mdm2-p53 Pathway

The present inventors identified a change in SRF-Enigma-Mdm2-p53 by aWestern blotting after injecting each of Ad-p53 or Ad-LacZ virus (5×10⁸pfu) into tail vein of mice (Balb/c, Female, Taconic) and after 24 hoursinjecting HGF (100 μg/kg) into tail vein of mice again, removing liverstime-wise. As the result, HGF induced protein expression bytranscriptionally activating SRF, Enigma was induced by SRF attranscription level, and an amount of protein increased. According to anincrease in Enigma, the amount of protein in Mdm2 increased andsimultaneously that of p53 decreased (FIGS. 10F and 10G).

The present inventors, to observe a change in Mdm2-p53 according to adirect change in an amount of Enigma expression, identified an amount ofeach protein by a Western blotting after injecting or not injectingAd-p53 into tail vein of mice (Balb/c, Female, Taconic) and after 24hours injecting Ad-F-Enigma, Ad-LacZ, PBS into tail vein of mice again,removing livers after 48 hours. Furthermore, after injectingAd-siEnigma, Ad-siControl and PBS into tail vein and injecting HGF intotail vein 8 hours before liver removal. As the result, if Enigmaexpression was high, Mdm2 was stabilized and p53 decreased; when Enigmaexpression was inhibited, Mdm2 stabilization by HGF was inhibited andp53 increased (FIG. 10H). The result suggests that SRF-Enigma-Mdm2-p53is present in vivo.

EXAMPLE 6 SRF and Enigma are Co-Expressed with Mdm2 in Human CancerTissues

The present inventors, to identify whether in human cancer tissues(stomach cancer: Chungnam National University Medical School, SouthKorea; liver cancer: Chonbuk National University Medical School andKeimyung University Medical School, South Korea) induction of Enigma bySRF may cause Mdm2 stabilization and then induce an decrease in p53,lysed liver cancer tissues, stomach cancer tissues and correspondingnormal tissues in tissue lysis buffer (Intron, Korea) and investigatedthem by a Western blotting. As the result, SRF and Enigma wereco-expressed with Mdm2 in ten cases of liver and stomach tumors, wherep53 was not detected (FIG. 13 a, indicated by a single asterisk).Furthermore, in tissue array (available on the World Wide Web attissuearray.com) planted in various types of human cancer tissues,Enigma-Mdm2 expression was observed by using an immuno-fluorescentstaining method. As the result, Enigma is co-localized with Mdm2 at anapical region of cytoplasm in stomach cancer and colon cancer tissues(FIG. 13B). The result suggests that SRF-Enigma-Mdm2 is activated andp53 is likely attenuated in rapidly proliferating cancer cells.

Example 7 Enigma Promotes Cell Viability and Confers Chemo-Resistance

The present inventors investigated whether Enigma decreases proteinlevel of p53, which induces apoptosis, and thereby increases cellsurvival ability and induces resistance to anti-cancer agents.

<7-1> p53-Dependent Induction of Apoptosis by Expression Inhibition ofEnigma

The present inventors identified whether Enigma's effect on cellsurvival is p53-dependent by cell counting method and crystal violetmethod after inducing Flag-Enigma(5 μg) or siEnigma (10 μg) into HCT116colon cancer cell lines of p53^(+/+) or p53^(−/−), and processing withadriamycin (ADR 20 μg/ml). As the result, cell's ability to surviveincreased in case of high Enigma expression in cells with wild-type p53alone; in case of inhibited Enigma expression the cell's ability tosurvive decreased (FIGS. 14A and 14B). Furthermore, in H1299, H358, PC3,and Hep3B cell lines where p53 is not expressed an decrease in Enigmaexpression had little effect on the cell's ability to survive (FIG. 15).The present inventors tested whether Enigma's effect on cell survival isa regulation effect on apoptosis by the TUNEL method and cell cycleanalysis. As the result, revealed is that in cell lines with wild-typep53 apoptosis by inhibition of Enigma expression was effective (FIG.14D). Thus, found is that in the presence of ADR, an increasing effectby Enigma of the cell's ability to survive is mediated by p53 and causedby inhibition of inducing apoptosis.

Also, the present inventors, to identify a change in Enigma-Mdm2-p53pathway in the presence of ADR, performed a Western blotting byprocessing ADR (2 μg/ml) under the same conditions above. As the result,irrespective of p53's presence, an decrease in Mdm2 by ADR was inhibitedby overexpression of Enigma, and an increase or decrease in Mdm2-p53 wasEnigma expression-dependent (FIG. 14C). Thus, found is that Enigmadecreases p53 level via stabilization of Mdm2 and thereby promotesresistance of cancer cells to ADR.

EXAMPLE 8 Enigma Confers Chemo-Resistance In Vivo

The present inventors identified that p53-mediated apoptosis isregulated dependently on Enigma expression, and investigated whether ADRresistance is induced by Enigma in mouse xenograft model made of HLK3, aliver cancer cell line.

<8-1> Enigma's Effect on Tumor Cell Viability

The present inventors investigated cell viability by the cell countingmethod by introducing F-Enigma (5 μg) or siEnigma (10 μg) into an HLK3cell line, untreated with ADR (20 μg/ml). As the result, in case of highEnigma expression, cell suicide by anti-cancer agents was inhibited; incase of inhibited Enigma expression, even in the absence of ADR, cellviability was effectively decreased (FIG. 16A and FIG. 17). The presentinventors, to identify a change in Enigma-Mdm2-p53 pathway in processwith ADR, performed a Western blotting in process with ADR (2 μg/ml)under the same conditions above. As the result, a chance in Mdm2-p53expression was Enigma expression-dependent (FIG. 16B).

<8-2> Enigma Confers Resistance to Etoposide and Nutlin3a

Although etoposide (ETC) and nutlin3a (Sigma, USA) have distinctmechanisms, both are anti-cancer agents to activate p53 and therebyinduce suicide of cancer cells (Science 303, 844-848, 2004). The presentinventors tested whether Enigma enables cancer cells to show resistanceto them. The present inventors identified a number of survived cells bythe cell counting method by processing ETC and nutlin3a respectively inan HLK3 cell, where F-Enigma (5 μg) is induced. As the result, thepresent inventors identified that, in case of Enigma overexpression,resistance to ETC and nutlin3a was induced (FIG. 16C). The presentinventors, to identify a change in Enigma-Mdm2-p53 pathway in processwith ADR, performed a Western blotting in process with ADR (2 μg/ml)under the same conditions above. As the result, a chance in Mdm2-p53expression was Enigma expression-dependent (FIG. 16B).

<8-3> Enigma Exhibits Resistance to ADR In Vivo

The present inventors, to test whether Enigma induces resistance oftumor cells to ADR in vivo, generated tumor on nude mice (Balb/c nu,Female, SLC, Japan). After first infecting Ad-F-Enigma (100 MOI),Ad-LacZ (100 MOI), Ad-siEnigma (200 MOI), Ad-siControl (200 MOI) into anHLK cell, the present inventors injected them into said mice(n=5/experimental group) subcutaneously. After 14 days when ADR (4mg/kg) was injected into tail vein, the size of tumor was observed for28 days. As the result, cancer cells where F-Enigma is expressed showedresistance to ADR, in proliferating similarly to control group untreatedwith ADR, and in a group where Enigma expression is inhibited,proliferation of cancer cells is most inhibited (FIGS. 16E and 16F). Thepresent inventors, to identify an expression change in Mdm2-p53 withincells forming a mass, identified expression aspect of each protein by aWestern blotting after, under the same conditions above, generating amass on said mice, processing it with ADR, and obtaining a cell lysissolution by separating the mass from the mice after 3 days. As theresult, in the presence of resistance to anti-cancer agents, Mdm2 wasincreased and p53 level decreased (FIG. 16G).

As described above, the present invention may be utilized for studies ona mechanism by which Enigma regulates protein levels of Mdm2 and p53through the ubiquitin-proteasome pathway, and on a mechanism by whichSRF-Enigma-Mdm2-p53 pathway is regulated in cancer cells. Furthermore,it may be utilized for developing anti-cancer agents via expressionregulation of Enigma, for developing a screening method of anti-canceragents by using Enigma expression or Enigma-Mdm2 interaction, and fordeveloping a method of assessing the prognosis of treatment withanti-cancer agents by using Enigma expression.

As aforementioned, the Enigma overexpression causes Mdm2 stabilizationand a decrease in p53, which was clearly caused by inhibition ofself-ubiquitination of Mdm2 and an increase in p53 ubiquitination. Thus,it can be known that cancer cells are effectively apoptosized byinhibiting Enigma expression, that the prognosis of anti-cancer therapymay be assessed by measuring Enigma expression from cancer patients, andthat substances showing anti-cancer activity may be screened byidentifying a specific interaction between Enigma and Mdm2, therebyselecting substances inhibiting the interaction, thereby weakening Mdm2stabilization and increasing p53 activity.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

What is claimed is:
 1. A method of screening candidate substances for ananti-cancer agent, the method comprising: 1) contacting PDZ and LIMdomain 7 (PDLIM7) and Mdm2 in the presence or absence of a candidatesubstance, wherein the candidate substance is a peptide, a protein, anantibody, a small molecule, or a natural substance; 2) measuring abinding level for PDLIM7 and Mdm2; and 3) selecting a candidatesubstance that decreases the binding level for PDLIM7 and Mdm2, comparedto in the absence of the candidate substance, thereby identifying ananti-cancer agent, wherein the cancer is liver cancer, stomach cancer,or colorectal cancer.
 2. A method of screening candidate substances foran anti-cancer agent, the method comprising: 1) bringing cellsexpressing PDZ and LIM domain 7 (PDLIM7) and Mdm2 into contact with acandidate substance; 2) measuring a binding level for PDLIM7 and Mdm2;and 3) selecting a candidate substance that decreases the binding levelfor PDLIM7 and Mdm2, compared to a control group untreated with thecandidate substance, thereby identifying an anti-cancer agent, whereinthe cancer is liver cancer, stomach cancer, or colon cancer.
 3. A methodof screening candidate substances for an anti-cancer agent, the methodcomprising: 1) treating cells expressing PDZ and LIM domain 7 (PDLIM7)and Mdm2 with a candidate substance, wherein the candidate substance isa peptide, a protein, or an antibody; 2) measuring a level of PDLIM7 orMdm2 protein; and 3) selecting a candidate substance that decreasesstability of PDLIM7 or Mdm2 protein, compared to a control groupuntreated with the candidate substance, thereby identifying ananti-cancer agent, wherein the cancer is liver cancer, stomach cancer,or colon cancer.
 4. The method of claim 3, wherein: the cells expressingPDLIM7 and Mdm2 also express Serum Response Factor (SRF) and wherein thecandidate substance further decreases stability of SRF, compared to acontrol group untreated with the candidate substance.